Aglucone isoflavone enriched vegetable protein concentrate and process for producing

ABSTRACT

Aglucone isoflavone enriched protein concentrates and processes for producing and recovering are disclosed. The process comprises an isoelectrie wash of a vegetable protein material to provide a protein concentrate, which is slurried and reacted with a sufficient amount of beta-glucosidase enzyme or esterase enzyme for a time period, temperature, and pH sufficient to convert at least a majority of the glucone isoflavones contained in the concentrate to aglucone isoflavones.

This is a continuation-in-part of U.S. patent application Ser. No.08/135,194, filed Oct. 12, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the production of an agluconeisoflavone enriched vegetable protein concentrate, by washing avegetable protein material to produce a vegetable protein concentrateand treating with one or more beta-glucosidase enzymes under conditionssuch that a majority of the glucone isoflavones are convened to agluconeisoflavones which are retained in the enriched protein concentrate.

BACKGROUND OF THE INVENTION

Isoflavones occur in a variety of leguminous plants, including vegetableprotein materials such as soybeans. These compounds include daidzin,6"-OAc daidzin, 6"-OMal daidzin, daidzein, genistin, 6"-OAc genistin,6"-OMal genistin, genistein, glycitin, 6"-OAc glycitin, 6"-OMalglycitin, glycitein, biochanin A, formononetin, and coumestrol.Typically these compounds are associated with the inherent, bitterflavor of soybeans, and in the production of commercial products, suchas isolates and concentrates, the focus has been to remove thesematerials. For example, in a conventional process for the production ofa soy protein isolate, in which soy flakes are extracted with an aqueousalkaline medium, much of the isoflavones are solubilized in the extract,and remains solubilized in the whey, which is usually discardedfollowing acid precipitation of the protein to form an isolate. Residualisoflavones left in the acid precipitated protein isolate are usuallyremoved by exhaustive washing of the isolate.

It has been recently recognized that the isoflavones contained invegetable proteins such as soybeans may inhibit the growth of humancancer cells, such as breast cancer cells and prostate cancer cells asdescribed in the following articles: "Genistein Inhibition of the Growthof Human Breast Cancer Cells, Independence from Estrogen Receptors andthe Multi-Drug Resistance Gene" by Peterson and Barnes, Biochemical andBiophysical Research, Communications, Vol. 179, No. 1, pp. 661-667, Aug.30, 1991; "Genistein and Biochanin A Inhibit the Growth of HumanProstate Cancer Cells but not Epidermal Growth Factor Receptor TyrosineAuto-phosphorylation" by Peterson and Barnes, The Prostate, Vol. 22, pp.335-345 (1993); and "Soybeans Inhibit Mammary Tumors in Models of BreastCancer" by Barnes, et al. Mutagens and Carcinogens in the Diet, pp.239-253 (1990).

Of the above isoflavones, several exist as glucosides, or as glucones,with a glucose molecule attached, at the seven position as illustratedin the formula below. Several of the glucones such as the 6"-OAcgenistin, contain an acetate group attached to the six position of theglucose molecule itself. While all of the isoflavones, including theglucosides are of interest in medical evaluation, the specificisoflavones of most interest are the aglucones, wherein the glucosemolecule is not attached. These isoflavones are not as water soluble asthe glucones or glucosides. Specific isoflavones in this category aredaidzein, genistein, and glycitein. These aglucones have the followinggeneral formula: ##STR1## wherein, R₁, R₂, R₃ and R₄ may be selectedfrom the group consisting of H, OH and OCH₃. It is therefore to theaglucones and enrichment of a vegetable protein concentrate with thesematerials to which the present invention is directed.

Methods are known in the art for converting glucone isoflavones toaglucone isoflavones, such as described in Japanese Patent Application258,669 to Obata et al. Such processes achieve only a moderate extent ofconversion and so are not desirable, particularly for large scalecommercial operations. In addition, known processes such as described inthe '669 application teach removing the isoflavones from the proteinmaterial and do not describe how to prepare an aglucone isoflavoneenriched protein concentrate. Thus, there is a need for a process ofconverting at least a majority and preferably substantially all gluconeisoflavones to aglucone isoflavones, and for producing an agluconeisoflavone enriched vegetable protein concentrate.

It is therefore an object of the present invention to provide anaglucone isoflavone enriched protein concentrate and a process forproducing the same. This, and other objects, are specifically achievedin the detailed description of the present invention set forth below.

SUMMARY OF THE INVENTION

The present invention provides an aglucone isoflavone enriched vegetableprotein concentrate and process for producing such. The methods forproducing such comprise washing a vegetable protein material comprisingglucone isoflavones with an aqueous solvent having a pH at about theisoelectric point of the protein material to produce a vegetable proteinconcentrate. The concentrate is then reacted with a sufficient amount ofone or more beta-glucosidase enzymes for a time period, temperature, andpH sufficient to convert at least a majority of the glucone isoflavonesin the concentrate to aglucone isoflavones, and thereby produce anaglucone isoflavone enriched protein concentrate. The present inventionalso provides methods for producing such concentrates whereinsupplemental beta-glucosidase is added to the wash or concentrate. Theresulting aglucone enriched concentrate can then be separated anddewatered. In addition, the present invention provides methods ofrecovering, in relatively high proportions, isoflavones in proteinconcentrates, from vegetable protein materials.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although the present invention will be described with respect to soybeanproducts and although the process is particularly suited for theproduction of aglucone isoflavone enriched concentrated soybeanmaterials, nevertheless the process is generally applicable to theproduction of an aglucone isoflavone enriched concentrate from a varietyof vegetable protein sources which contain isoflavones. An example of asuitable vegetable protein material is a soy material, a soybeanmaterial, or a vegetable protein material comprising soybeans. The term"soybean material" as used herein refers to soybeans or any soybeanderivative.

The starting material in accordance with the preferred embodimentconcentrate is soybean flakes, from which the oil has been removed bysolvent extraction. Typically, a soy protein concentrate is producedfrom soybean flakes by washing the flakes with an aqueous solvent havinga pH at about the isoelectric point of the protein, which in the case ofsoy protein is about 4.4 to 4.6. An edible acid is typically added tothe water to provide an isoelectric wash for the soy protein material.The isoelectric wash removes a large mount of water solublecarbohydrates and other components, but removes little of the protein tothereby provide a protein concentrate, which will typically have aprotein content on a dry basis of about 60-75% by weight. The gluconeisoflavones contained in the vegetable protein or soy material areremoved by isoelectric washing. But because of the relatively low pH,the mounts removed are less than in higher pH extractions. Therefore, itis preferred for the purposes of the preferred embodiment and insofar asmaximizing recovery of the isoflavones that isoelectric washing belimited to a single step or at most one additional wash be carried out,also at the isoelectric point. It is also preferred that the weightratio of aqueous solvent used to wash the protein material relative tothe mount of protein material is about 5:1 to about 10:1.

The resulting concentrate is suspended in water at a solids level ofabout 10 to about 15% by weight and then reacted with one or morebeta-glucosidase enzymes to convert at least a majority and preferablysubstantially all the glucone isoflavone material contained in theconcentrate to aglucone isoflavones. The optimum pH range for thebeta-glucosidase enzyme will vary depending on the specificbeta-glucosidase enzyme used, but typically will vary between about 4and about 8. The pH of the extract is typically adjusted to about the pHrange at which the specific enzyme is most active prior to reaction withthe enzyme. The pH is typically adjusted by the addition of an edibleacid, such as acetic, sulfuric, phosphoric, hydrochloric, or any othersuitable reagent.

The beta-glucosidase enzyme may be naturally present in the soybeanmaterial or present from microbial growth, referred to herein as"residual" enzyme, or may be added to the concentrate. Added enzyme isreferred to herein as "supplemental enzyme". Generally, if theconcentration of residual enzyme in the concentrate is insufficient toconvert a majority, and preferably substantially all, the isoflavones inglucone form to aglucone form, then supplemental enzyme should be added.The amount of enzyme sufficient to perform the conversion of isoflavonesvaries upon a multitude of factors, including the type of enzymespresent, distribution of enzyme concentrations, pH of the system, andactivities of enzymes present. Once sufficient concentrations of enzymesare present, either via residual enzymes, supplemental enzymes, or both,the concentrate is reacted with the beta-glucosidase enzymes for a timeperiod, temperature, and pH sufficient to convert at least a majorityand preferably substantially all the glucone isoflavones contained inthe concentrate to the aglucone form.

Preferred supplemental beta-glucosidase enzymes include Biopectinase100L and 300L, Biopectinase OK 70L, Lactase F, and Lactozyme. Lactase Fis available from Amano International Enzyme Co., Inc., P.O. Box 1000,Troy, Va. 22974, which has an optimum pH range of about 4-6, andLactozyme is available from Novo Industries, Enzyme Division, Novo Alle,DK-2880 Bagsvaerd, Denmark, which has an optimum pH range of about 7.Biopectinase 100L, Biopectinase 300L, and Biopectinase OK 70L areavailable from Quest International, Sarasota, Fla. Supplemental enzymesare added in amounts sufficient to convert at least a majority andpreferably substantially all the glucone isoflavones to aglucones. Ininstances where it is necessary to add supplemental enzymes, the mountof enzyme added is about 0.5% to about 5% by weight of the proteinconcentrate on a dry basis.

Another class of enzymes suitable for administration as supplementalenzymes are esterase enzymes. These enzymes are believed to be wellsuited to the preferred embodiment processes described herein as theyconvert the acetate and malonate conjugates to glucone isoflavones byremoving the acetate and malonate groups from the isoflavone conjugates.In the most preferred embodiment, both types of enzymes,beta-glucosidase and esterase enzymes are utilized.

The processes of the preferred embodiment are preferably one-stepprocesses and achieve very high degrees of conversion of isoflavones(from glucone form to aglucone form), in relatively short time periods,and with relative ease and economy. The term "one-step" reaction processas used herein refers to a reaction process in which certain processparameter values are generally maintained over the course of thereaction process. These process parameters include pH and temperature.

The very high degrees of conversion are such that at least a majority,and preferably, substantially all the isoflavones in glucone formpresent in the concentrate are convened to aglucone form. The term "amajority" refers to extent of conversion of glucone isoflavones toaglucone isoflavones of at least about 50%. The term "substantially all"refers to extent of conversion of glucone isoflavones to agluconeisoflavones of at least about 80%, and most preferably at least about90%.

Although not wishing to be bound to any particular theory, it isbelieved that the surprisingly and unexpectedly high degrees ofconversion of the processes described here in result from a combinationof process parameters utilized during the one-step reaction process. Itis preferred that pH of the reaction system be maintained, orapproximately so, at a value of about 4 to about 8, and most preferablyat a value at which the enzyme(s) are most active prior to reaction withthe isoflavone conjugate(s) during the one-step reaction process. It ispreferred that the temperature of the reaction system be maintained, orapproximately so, at a temperature of from about 40° C. to about 60° C.,and most preferably at a temperature of about 60° C. during the one-stepreaction process. Generally, the time periods necessary to achieveconversion of substantially all glucone isoflavones to aglucones via theone-step processes described herein are from about 2 hours to about 24hours.

An alternative procedure for providing an aglucone enriched concentrateis to combine isoelectric washing and reaction with the beta-glucosidaseenzymes into a single step, wherein the soy starting material issuspended in the isoelectric wash and one or more beta-glucosidaseenzymes having an optimum pH at about the isoelectric point such asLactase F described above is added directly to the slurry. Reaction isthen carried out under the previously described general conditions toconvert at least a majority and preferably substantially all the gluconeisoflavones to aglucones. This represents a preferred and simplifiedprocess without first washing the material at the isoelectric point toremove soluble carbohydrates and thereby avoid any possible loss of theisoflavones by washing.

The protein concentrate can then be dewatered by conventional proceduresincluding centrifugation and drying techniques to provide a concentratehaving a genistein level on a dry basis of about 1.0 to about 2.0mg/gram and a daidzein level on a dry basis of about 0.7 to about 1.5mg/gram.

The present invention also provides methods of recovering isoflavones ina protein concentrate, in very high proportions, from a vegetableprotein material such as a soybean material. The recovery levelsobtainable by the processes described herein are typically at least 50%,preferably 65%, and most preferably 80%, based upon the total of allforms of the particular isoflavone in the starting vegetable proteinmaterial. Although not wishing to be bound to any particular theory, itis believed that the high recoveries stem from the conversion reactionsdescribed herein coupled with the various processing operations alsodescribed. By converting glucone isoflavone conjugates, which arerelatively soluble, to less soluble aglucone forms, at a particularstage of processing, it is possible to recover in the resulting product,a high percentage of the isoflavones from the feed material.

The following examples describe specific but non-limiting embodiments ofthe present invention.

EXPERIMENTAL

A mixture of 15 grams soybean meal and 150 grams of water was adjustedto a pH of 7. 1.5 grams of Biopectinase 100L was added to the mixture.The mixture was incubated at 60° C. for two hours at which time the pHwas adjusted to 4.5. An additional 1 gram of Biopectinase 100L was addedand the resulting mixture was incubated for two hours. After incubation,the resulting aglucone isoflavone enriched protein/fiber concentrate wasrecovered. Recovery of isoflavones in the protein/fiber concentrate isreported in Table 1 set forth below.

                                      TABLE 1                                     __________________________________________________________________________                      6"-OMAL-                                                                             6"-OAC-             6"-OMAL-                                    GENISTIN                                                                             GENISTIN                                                                             GENISTIN                                                                             GENISTEIN                                                                            DAIDZIN                                                                             DAIDZIN                          SAMPLE     %      %      %      %      %     %                                __________________________________________________________________________    One-Step Conversion                                                           Starting material                                                                        48     49     0       3     47    47                               Protein/Fiber Conc.                                                                       8     27     0      66      7    23                               __________________________________________________________________________                     6"-OAC-             6"-OMAL-                                                  DAIDZIN                                                                             DAIDZEIN                                                                             GLYCITIN                                                                             GLYCITIN                                                                             GLYCITEIN                         SAMPLE           %     %      %      %      %                                 __________________________________________________________________________    One-Step Conversion                                                           Starting material                                                                              1      4     43     41     16                                Protein/Fiber Conc.                                                                            0     70     50     15     35                                __________________________________________________________________________

These data indicate the degree of conversion attainable by a combinationof residual enzyme(s) and supplemental enzyme. Significant conversion ofisoflavone conjugates to aglucones occurred, particularly for genisteinand daidzein. The concentration of each type of isoflavone describedherein is based upon the total of all forms of that isoflavone type.

In another series of experiments, the percent recovery of genistein anddaidzein in a protein concentrate derived from soybeans wasinvestigated. The percent recovery was found by determining the amountof genistein (or daidzein) in the isolate, and expressing that amount asa percentage based upon the total amount of all forms of genistein (ordaidzein) in the soybean starting material. 100 g of defatted soy flourwas added to 1600 g of water which had been adjusted to a pH of 4.5 bythe addition of hydrochloric acid. The slurry was heated to 50° C., and2% by dry weight of the curd of an enzyme having beta-glucosidaseactivity, specifically Lactase F, was added. The slurry was allowed toreact for 16 hours at 50° C. to ensure complete conversion of theglucone isoflavones to the aglucone form. The protein concentrate wasseparated from the aqueous solvent by centrifugation to form an agluconeenriched concentrate. Further washing of the concentrate was avoided.The amount of genistein recovered in the isolate was 82% of the total ofall forms of genistin and genistein in the starting soybean material(defatted soy flour). Similarly, the amount of daidzein recovered in theisolate was 64%.

The following is a description of a method for quantifying isoflavonesin soy products. The isoflavones are extracted from soy products bymixing 0.75 gram of sample (spray dried or freely ground powder) with 50ml of 80/20 methanol/water solvent. The mixture is shaken for 2 hours atroom temperature with an orbital shaker. After 2 hours, the remainingundissolved materials are removed by fitration through Whatman No. 42filter paper. Five ml of the filtrate are diluted with 4 ml of water and1 ml of methanol.

The extracted isoflavones are separated by HPLC (High Performance LiquidChromatography) using a Beckman C18 reverse phase column. Theisoflavones are injected on to the column and eluted with a solventgradient starting with 88% methanol, 10% water, and 2% glacial aceticacid and ending with 98% methanol and 2% glacial acetic acid. At a flowrate of 0.4 ml/min, all the isoflavones--genistin, 6"-0-Acetylgenistin,6"-0-Malonylgenistin, genistein, daidzin, 6"-0-Acetyldaidzin,6"-0-Malonyldaidzin, daidzin, glycitin and its derivatives andglycitein--are clearly resolved. Peak detection is by UV absorbance at262 mm. Identification of the peaks was by mass spectrometer.

Quantification is achieved by using pure standards (genistin, genistein,daidzin and daidzein) purchased from Indofine Chemical Company,Sommerville, N.J. Response factors (Integrated area/concentration) arecalculated for each of the above compounds and are used to quantitateunknown samples. For the conjugated forms for which no pure standardsare available, response factors are assumed to be that of the parentmolecule but corrected for molecular weight difference. The responsefactor for glycitin is assumed to be that for genistin corrected formolecular weight difference.

This method provides the quantities of each individual isoflavone. Forconvenience, total genistein, total daidzein and total glycitein can becalculated from the above data and represent the aggregate weight ofthese compounds if all the conjugated forms are converted to theirrespective unconjugated forms. These totals can also be measureddirectly by a method using acid hydrolysis to convert the conjugatedforms.

Of course, it is understood that the foregoing are merely preferredembodiments of the invention and that various changes and alterationscan be made without departing from the spirit and broader aspectsthereof as set forth in the appended claims, which are to be interpretedin accordance with the principals of patent law including the Doctrineof Equivalents.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A process for producingan aglucone isoflavone enriched protein concentrate from a vegetableprotein material comprising:(a) washing a vegetable protein materialcomprising glueone isoflavones with an aqueous solvent having a pH atabout the isoelectric point of said vegetable protein material toproduce a vegetable protein concentrate comprising glucone isoflavones;and (b) reacting said glucone isoflavones with a sufficient amount ofenzyme which is at least one of beta-glucosidase enzyme and esteraseenzyme for a time period, temperature, and pH sufficient to convert atleast a majority of said glucone isoflavones in said concentrate toaglucone isoflavones, and thereby produce an aglucone isoflavoneenriched protein concentrate.
 2. The process as set forth in claim 1wherein said time period is from about 2 hours to about 24 hours.
 3. Theprocess as set forth in claim 2 whereto said time period is about 24hours.
 4. The process as set forth in claim 1 wherein said temperatureis about 40° C. to about 60° C.
 5. The process as set forth in claim 4wherein said temperature is about 60° C.
 6. The process as set forth inclaim 1 wherein the pH of the reacting step is from about 4 to about 8.7. The process as set forth in claim 6 wherein the pH of the reactingstep is about 4.5.
 8. The process as set forth in claim 1 wherein saidtime period is about 24 hours, said temperature is about 60° C., and thepH of the reacting step is about 4.5.
 9. The process as set forth inclaim 1 wherein the steps of washing said vegetable protein material andreacting said glucone isoflavones with said beta glucosidase enzyme isperformed in one operation.
 10. The aglucone isoflavone enriched proteinconcentrate produced by the method of claim
 1. 11. The process as setforth in claim 1 wherein said aglucone isoflavone enriched proteinconcentrate is made from soybeans.
 12. The process as set forth in claim1 wherein said vegetable protein material comprises a soybean material.13. The process as set forth in claim 1 wherein substantially allglucone isoflavones are converted to aglucone isoflavones.
 14. A processfor producing an aglucone isoflavone enriched protein concentrate from avegetable protein material comprising:(a) washing a vegetable proteinmaterial comprising glucone isoflavones and sufficient residual enzymewhich is at least one of beta-glucosidase enzyme or esterase enzyme,with an aqueous solvent having a pH at about the isoelectric point ofsaid vegetable protein material to produce a vegetable proteinconcentrate comprising glucone isoflavones; and (b) reacting saidglucone isoflavones with said residual enzyme for a time period,temperature, and pH sufficient to convert at least a majority of saidglucone isoflavones in said concentrate to aglucone isoflavones, andthereby produce an aglucone isoflavone enriched protein concentrate. 15.The process as set forth in claim 14 wherein said time period is fromabout 2 hours to about 24 hours.
 16. The process as set forth in claim15 wherein said time period is about 24 hours.
 17. The process as setforth in claim 14 wherein said temperature is about 40° C. to about 60°C.
 18. The process as set forth in claim 17 wherein said temperature isabout 60° C.
 19. The process as set forth in claim 14 wherein the pH ofthe reacting step is from about 4 to about
 8. 20. The process as setforth in claim 19 wherein the pH of the reacting step is about 4.5. 21.The process as set forth in claim 14 wherein said time period is about24 hours, temperature is about 60° C., and the pH of the reacting stepis about 4.5.
 22. The process as set forth in claim 14 wherein the stepsof washing said vegetable protein material and reacting said gluconeisoflavones with said beta glucosidase enzyme are performed in oneoperation.
 23. The aglucone isoflavone enriched protein concentrateproduced by the method of claim
 14. 24. The process as set forth inclaim 14 wherein said concentrate is made from soybeans.
 25. The processas set forth in claim 14 wherein said vegetable protein materialcomprises a soybean material.
 26. The process as set forth in claim 14wherein substantially all glucone isoflavones are converted to agluconeisoflavones.
 27. A process for producing an aglucone isoflavone enrichedprotein concentrate from a vegetable protein material comprising:(a)washing a vegetable protein material comprising glucone isoflavones withan aqueous solvent having a pH at about the isoelectric point of saidvegetable protein material to produce a vegetable protein concentratecomprising glucone isoflavones; (b) adding supplemental enzyme which isat least one of beta-glucosidase enzyme and esterase enzyme to saidconcentrate so that the total concentration of enzyme in saidconcentrate is sufficient to convert at least a majority of said gluconeisoflavones in said concentrate to aglucone isoflavones; and (c)reacting said glucone isoflavones with said enzyme for a time period,temperature, and pH sufficient to convert at least a majority of saidglucone isoflavones in said concentrate to aglucone isoflavones, andthereby produce an aglucone isoflavone enriched concentrate.
 28. Theprocess as set forth in claim 27 wherein said time period is from about2 hours to about 24 hours.
 29. The process as set forth in claim 28wherein said time period is about 24 hours.
 30. The process as set forthin claim 27 wherein said temperature is about 40° C. to about 60° C. 31.The process as set forth in claim 30 wherein said temperature is about60° C.
 32. The process as set forth in claim 27 wherein the pH of thereacting step is from about 4 to about
 8. 33. The process as set forthin claim 32 wherein the pH of the reacting step is about 4.5.
 34. Theprocess as set forth in claim 27 wherein said time period is about 24hours, said temperature is about 60° C., and the pH of the reacting stepis about 4.5.
 35. The process as set forth in claim 27 wherein the stepsof washing said vegetable protein material and reacting said gluconeisoflavones with said beta glucosidase enzyme are performed in oneoperation.
 36. The aglucone isoflavone enriched concentrate produced bythe method of claim
 27. 37. The process as set forth in claim 27 whereinsaid concentrate is made from soybeans.
 38. The process as set forth inclaim 27 wherein said vegetable protein material comprise a soybeanmaterial.
 39. The process as set forth in claim 27 wherein substantiallyall glucone isoflavones are converted to aglucone isoflavones.
 40. Anaglucone isoflavone enriched protein concentrate having on a dry basis agenistein content of about 1.0 to about 2.0 mg/gram and a daidzeincontent of about 0.7 to about 1.5 mg/gram.
 41. The process as set forthin claim 1 wherein the pH of the reacting step is at a value at whichsaid enzyme is most active prior to reaction with said isoflavones. 42.The process as set forth in claim 14 wherein the pH of the reacting stepis at a value at which said residual enzyme is most active prior toreaction with said isoflavones.
 43. The process as set forth in claim 27wherein the pH of the reacting step is at a value at which saidsupplemental enzyme is most active prior to reaction with saidisoflavones.
 44. A process for recovering in a protein concentrate, atleast 50% of an isoflavone in a vegetable protein materialcomprising:(a) washing a yegetable protein material comprisingisoflavones with an aqueous solvent having a pH at about the isoelectricpoint of said vegetable protein material to produce a vegetable proteinconcentrate comprising said isoflavones; (b) reacting said isoflavoneswith a sufficient amount of an enzyme for a time period, temperature,and pH sufficient to convert at least a majority of said isoflavones insaid concentrate to less soluble isoflavones, and thereby produce anisoflavone enriched protein concentrate containing at least 50% of theisoflavones in said vegetable protein material.
 45. The process as setforth in claim 44 wherein said concentrate contains at least 65% of theisoflavones in said vegetable protein material.
 46. The process as setforth in claim 44 wherein said concentrate contains at least 80% of theisoflavones in said vegetable protein material.
 47. The concentrateproduced by the method of claim
 44. 48. The concentrate produced by themethod of claim
 45. 49. The concentrate produced by the method of claim46.
 50. The process as set forth in claim 44 wherein said vegetableprotein material comprises a soybean material.
 51. The process as setforth in claim 44 wherein said enzyme is selected from the groupconsisting of beta-glucosidase enzyme and esterase enzyme.